In the past 2 years working with DTM we have extensively modified the protocols used to prepare DCs at the NIH. In brief, we have shortened the period of monocyte incubation recombinant GM-CSF and IL4 to prepare cells from 4 days to 3 days, and modified the agents used to mature DCs from CD40L to LPS and IFN-g. These changes dramatically change the phenotype and IL12 producing capacity of the final DC product. Most recently, we have studied in greater detail how the timing of DC differentiation and maturation affects DC performance. These studies document 2 points: First, extending the incubation of DCs with GM-CSF/IL4 for more than 4 days adversely affects the abiility of DCs to make costimulatory molecules and IL12 after maturation. Second, we have established, that maturation of DCs with a mixture of LPS plus IFN-g prevents the undesirable downregulation of DC IL12 production noted using LPS alone or CD40L and IFN-g. The modified manufacturing methods we developed have been adopted by the cell processing section and will be used to prepare DCs in two upcoming clinical protocols initiated by Crystal MacKall and Alan Wayne. The resulting data will be very valuable in assessing the clincal efficacy of the revised DC product.